Tissue Engineering
Tissue engineering may be defined as the art of reconstructing or regenerating mammalian tissues, both structurally and functionally (Hunziker, Osteoarth. Cart. 10:432-63, 2002). Tissue engineering generally includes the delivery of a synthetic or natural scaffold that serves as an architectural support onto which cells may attach, proliferate, and synthesize new tissue to repair a wound or defect.
An example of a tissue that is prone to damage by disease and trauma is the articular cartilage, one of several types of cartilage in the body, found at the articular surfaces of bones. Damage to cartilage may result from an inflammatory disease such as rheumatoid arthritis, from a degenerative process such as osteoarthritis or from trauma such as intraarticular fracture or following ligament injuries. Cartilage lesions are often associated with pain and reduced function and generally do not heal. Without medical intervention, a patient may require total joint replacement.
Current therapeutic strategies for repairing damaged cartilage encompass procedures that induce a spontaneous repair response and those which reconstruct the tissue in a structural and functional manner. The former includes surgical techniques that expose the subchondral bone thereby allowing the infiltration of bone marrow progenitor cells to initiate the healing response. Often the induced tissue is of a mixed fibrocartilage type, is not durable and the clinical improvements are short lived. The latter strategy includes transplantation of chondral or osteochondral cells or tissue from an autologous or an allogeneic source. Autologous Chondrocyte Transplantation (ACT) relies on transplanting into a cartilage lesion autologous chondrocytes, which have been isolated from a patient's cartilage biopsy and expanded in vitro. In fact, this technique requires a complicated procedure involving two surgical sites and shows limited clinical success.
Matrices useful for tissue regeneration and/or as biocompatible implants useful for tissue culture are well known in the art. These matrices may therefore be considered as substrates for cell growth either in vitro or in vivo. Suitable matrices for tissue growth and/or regeneration include both biodegradable and biostable entities. Among the many candidates that may serve as useful matrices claimed to support tissue growth or regeneration are gels, foams, sheets, and porous structures of different forms and shapes.
Typical bioabsorbable materials for use in the fabrication of porous wound dressings or implants include both synthetic polymers and biopolymers such as structural proteins and polysaccharides. The biopolymers may be selected or manipulated to provide greater or lesser degrees of flexibility or susceptibility to degradation.
U.S. Pat. No. 5,607,474 discloses a molded biodegradable two-layer implant for repair of defects having two dissimilar tissue types. Each layer is prepared separately and subsequently joined together.
U.S. Pat. Nos. 6,306,424; 6,333,029 and 6,534,084 disclose a porous biocompatible foam prepared using a modified polymer-solvent phase separation technique that results in foam having a gradient in stiffness, flexibility, bioabsorption and or pore architecture, associated with a transition in composition. The disclosure teaches foams prepared from synthetic polymers such as aliphatic polyesters.
Fibrin
Fibrinogen is a major plasma protein, which participates in the blood coagulation process. Upon blood vessel injury, fibrinogen is converted to insoluble fibrin which serves as the scaffold for a blood clot. Fibrin is known in the art as a tissue adhesive medical device useful for wound healing and tissue repair. Lyophilized plasma-derived protein concentrate (comprising fibrinogen, Factor XIII and fibronectin), in the presence of calcium ions and the serine protease thrombin, forms an injectable biological sealant (fibrin glue). U.S. Pat. No. 5,411,885 discloses a method of embedding and culturing tissue employing fibrin glue.
The fibrin fiber size, density and rate of degradation of thrombin-induced fibrin gels are affected by several different factors. (Carr M E, Thromb. Haemost., 59(3)535-9, 1988; Carr M E and Alving B M, Blood Coag. Fibrinol. 6:567-73, 1995) The factors include fibrinogen source i.e. pure fibrinogen or plasma, fibrinogen, thrombin and factor XIII concentration, ion content, presence of fibronectin, calcium ions and dextran and other factors. In general, fibrin gels having thicker fibrin fibers, which result from low thrombin concentrations, low ionic strength, higher calcium or fibrinogen concentrations undergo fibrinolysis at a faster rate than fibrin gels having thinner fibers. A plasma protein gradient in a fibrin gel or in a porous, plasma protein matrix has not been taught.
U.S. Pat. No. 4,642,120 teaches the use of fibrinogen-containing glue in combination with autologous mesenchymal or chondrocytic cells to promote repair of cartilage and bone defects. U.S. Pat. No. 5,260,420 discloses a method for preparation and use of biological glue comprising plasma proteins for therapeutic use. U.S. Pat. No. 6,440,427 provides an adhesive composition consisting substantially of fibrin forming components and a viscosity-enhancing polysaccharide such as hyaluronic acid.
U.S. Pat. No. 5,972,385 discloses a lyophilized crosslinked collagen-polysaccharide matrix, with optional fibrin, that is administered per se or in combination with therapeutics for tissue repair. U.S. Pat. Nos. 5,206,023 and 5,368,858 disclose a method and composition for inducing cartilage repair comprising dressing the site with a biodegradable matrix formed by mixing matrix forming material with a proliferative agent and a transforming factor.
A fibrinogen-containing freeze-dried fleece-like structure for use as a wound dressing, filling for bone cavities or support material for release of active materials has been disclosed in U.S. Pat. No. 4,442,655. The structure is prepared by premixing fibrinogen and thrombin solutions, pouring into a mold, freezing and lyophilizing.
A freeze-dried fibrin web for wound healing has been disclosed in U.S. Pat. Nos. 6,310,267 and 6,486,377. The preparation of said web necessitates a single- or multi-stage dialysis of the fibrinogen solution. According to that disclosure, the single-stage or multistage dialysis of the fibrinogen solution changes crucially its composition by reducing the concentration of salts and amino acids. The dialysis is carried out in an aqueous solution of a physiologically compatible inorganic salt and an organic complexing agent.
U.S. Pat. No. 6,599,515 discloses a porous structure of fibrin or fibrinogen wherein the structure in its substantially dry form, has a compression strain of less than 8%, and a creep modulus higher than 1.5×106 Pa. The mechanical properties are obtained by polymerization of the fibrin or fibrinogen materials in the presence of an amount of a calcium-inhibiting agent, preferably an anticoagulant. After hydration, the structure has a porosity wherein at least 50% by volume of the total porosity is formed by channels with an open cross section of more than 500 μm2.
A storage stable fibrin sponge containing a blood clotting activator for hemostasis, tissue adhesion, wound healing and cell culture support is disclosed in U.S. Pat. No. 6,548,729. According to that disclosure, the restoration of moisture or water content following lyophilization is crucial for obtaining a soft, adaptable, absorbent sponge. The sponge may further be impregnated with additives such as a blood clotting activator, stabilizers, preservatives and other agents.
A freeze-dried fibrin clot for the slow release of an antibiotic is described by Itokazu (Itokazu et al., Infection 25:359-63, 1997).
U.S. Pat. Nos. 5,466,462 and 5,700,476 teach a bioresorbable heteromorphic sponge comprising a biopolymer matrix structure, at least one substructure and at least one pharmacologically active agent. The substructures allow the incorporation of one or more active agents into the final product for phasic release. U.S. Pat. No. 5,443,950 relates to the growth of cells derived from a desired tissue on a pre-established stromal support matrix. U.S. Pat. No. 5,842,477 discloses a method of in vivo cartilage repair by implanting a biocompatible, three-dimensional scaffold in combination with periosteal/perichondrial tissue and stromal cells, with or without bioactive agents. U.S. Pat. No. 6,569,172 discloses an implantable article for cartilage repair comprising a support matrix, and a mixture of chondrocyte cells and adhesive adhered to an edge of said support matrix.
PCT patent application WO 03/079985 teaches a method of preparing a biomimetic scaffold comprising the steps of providing two or more bio-ink solutions and co-depositing said bio-ink solutions to create the scaffold. Fibrinogen, thrombin and collagen are disclosed as examples of structural bio-inks. A scaffold having a patterned three-dimensional spatial and/or concentration gradient of therapeutic or structural elements is cited, yet a matrix having a plasma protein gradient is neither taught nor suggested.
PCT patent application WO 03/007873 by some of the applicants of the present invention discloses a fibrin matrix comprising plasma proteins and at least one anti-fibrinolytic agent, optionally further comprising agents such as polysaccharides, anionic polysaccharides, glycosaminoglycans, or synthetic polymers added in the preparation to improve certain physical, mechanical and biological properties of the matrix. Copending international patent application PCT/IL2004/000088 by some of the applicants of the present invention teaches a porous plasminogen-free plasma protein sponge and a method of preparing the sponge. The sponge may be prepared by sequential transferring of the thrombin solution and plasma protein solution into a mold or solid receptacle followed by freezing the clotted mixture and lyophilizing or alternatively, premixing the plasma protein solution with thrombin solution and casting into a mold or support prior to achieving clotting; the clotted mixture is frozen and lyophilized.
Collagen
Collagen is the most abundant protein in the body and constitutes a major part of the extracellular matrix. Collagen matrices and sponges useful for tissue regeneration are well known in the art. PCT publication WO 96/24310 discloses a multistage collagen based template or implant characterized by a first layer comprising a dense collagen membrane secured to a second layer comprising a porous collagen matrix.
U.S. Pat. No. 4,837,379 discloses a fibrin-collagen tissue equivalent comprising (i) a hydrated collagen lattice contracted by a contractile agent, such as fibroblasts, and (ii) fibrin. According to that patent, the tissue equivalents are prepared either by casting the collagen and fibrin lattice together or by incorporating the fibrin into the collagen lattice after the lattice is formed. Alternatively, a layered tissue equivalent may be formed.
A porous collagen structure impregnated with a slow setting fibrin adhesive at a fibrin adhesive to collagen volume proportion of at least 1 to 4, useful for osteocartilaginous reconstruction, has been disclosed in WO 93/16739. A method of producing a lyophilized tissue adhesive useful for wound healing based on collagen and fibrin is taught in U.S. Pat. No. 4,600,574. The method comprises the steps of (a) impregnating a tissue compatible flat material selected from collagen, gelatin and polysaccharide with a solution comprised of fibrinogen and factor XIII, and (b) lyophilizing said impregnated flat material to obtain a coherent matrix of said tissue-compatible flat material.
There remains an unmet need for a natural, three-dimensional matrix for use in tissue regeneration and repair that integrates a matrix having optimal pore size, pore distribution and interconnected channels for cell maintenance and nutrient diffusion while it provides a structural support.